JPH022373A - Gene coding aminopeptidase p, recombinant dna containing the same gene, viable cell strain containing the same recombinant dna and production of aminopeptidase p using the same cell - Google Patents

Abstract

PURPOSE:To produce aminopeptidase P using a microbial strain belonging to genus Escherichia or genus Saccharomyces by extracting a DNA having a genetic information participating in the production of aminopeptidase P and treating the DNA by a genetic engineering technique. CONSTITUTION:Escherichia coli HB101 is used as a DNA donor and a DNA having a genetic information participating in aminopeptidase formation is extracted from the strain. A microorganism of genus Escherichia or Saccharomyces is transformed with a plasmid integrated with said DNA. The above DNA has a base sequence starting from the 1st - 3rd ATG sequence and ending at the 1321st - 1323rd CAA sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing amine iguccigoose P by genetic recombination.

(Prior art) The presence of amino 4-detidase P has been recognized in Escherichia and Ivy kidney (
Yaron, A., & M1ynor+ D., B
ioehsm.

Blophyi, Rss, Commun, 32.
658 (1968), Dehm.

P., & Nordwig, Am., Eur., L.
Biochem, 17.364-371 (1970)
).

However, these conventionally known microorganisms or animal cells have extremely low production capacity for aminopeptidase-P, and are therefore unsuitable as an economical method for producing aminobutidase-P.

(Problem to be solved by the present invention) The problem to be solved by the present invention is to provide an economical method for producing amino 4-detidase P by microorganisms belonging to the genus Escherichia or Satucharomyces using genetic engineering techniques. It's about doing.

(Means for Solving the Problems) In order to solve the above-mentioned problems, the present inventors used Escherichia coli HBIOI bacteria as a deoxyribonucleic acid (hereinafter referred to as DNA) donor bacterium, and this produced diamino-4-butidase-P. DNA containing genetic information involved in the production of amino-(putidase P) is then obtained, and this cilasmid is introduced into a microorganism of the genus Escherichia or Satucharomyces. Among these, recombinant microorganisms with enhanced amino R-butidase P activity were selected.

It has been found that by using the microorganism of the present invention, aminopeptidase P can be produced at an extremely high yield compared to conventional methods using natural strains. That is, several microorganisms having aminopeptidase-P activity are known (T, Yoshimot).

at al,, Agrtc, Biol, Chsm
, 52.217 (1988)) From this, chromosomal DNA
Extract H, 5ait using a conventional method.

and K, Miura, Bioch4m, Bi
ophys, Acta, 72°619 (1963)
) and treated with restriction endonucleases in a conventional manner.

In our experiments, Pst was used as a restriction endonuclease.
DNA fragments carrying the genetic information involved in aminopeptidase-P production were obtained using I, but other restriction endonucleases such as BamH[, 5at1 + Hi
Pst III, EcoRI, etc.
Results similar to I may be obtained.

The vector DNA may be any one obtained from Escherichia coli relaxed type plasmids such as pBR322 and pUc19. No specific method is required to incorporate a DNA fragment carrying genetic information involved in aminopeptidase-P production into this vector.

All conventional transformation methods have not yet been used to infuse Escherichia microorganisms with cilasmids containing the DNA fragments that carry the genetic information involved in aminobezotidase P production. It is possible.

As recipient bacteria for the recombinant plasmid, it is better to use strains of the genus Escherichia that do not have or have weak aminobegutidase P activity, as they will not incorporate DNA fragments that carry the genetic information involved in aminobegutidase P production. This method is convenient for selecting and isolating transformed strains harboring plasmids.

The method for culturing the aminopeptidase-P producing bacteria thus obtained is not particularly different from the conventional culture method for Escherichia. That is, the culture medium is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins.

As the carbon source, glucose, sucrose, etc., starch hydrolysis containing these, molasses, etc. are used. Ammonia gas, aqueous ammonia, ammonium salts, and others can be used as the nitrogen source. More preferably, natural materials such as hard detone, tryptone, meat extract, and yeast extract are also used.

Cultivation is carried out under aerobic conditions, adjusting the culture medium and temperature as appropriate, until the production and accumulation of aminopeptidase-P is substantially at its maximum.

Amino from cultured bacteria! The following method is usually used to collect butidase P.

After collecting the cultured cells using a refrigerated centrifuge or the like, suspend the cells in an appropriate buffer and crush the cells using ultrasound or Dynomill to obtain an extract.

Amino! The bunadase P gene can also be expressed in yeast by incorporating it into a suitable expression vector and introducing it into a host cell. Several types of shuttle vectors for expressing foreign genes in yeast have already been reported. (Heitzman et al. Nature 293
, 717-722 (1981), Miyashita et al., Proc.
.

Natl, Acad, Set, USA, 80.
1-5 (1983)) These vectors can be propagated in both Escherichia coli and yeast hosts.

It also contains the yeast gene promoter sequence.

Basically all expression vectors can be used to express the aminobezotidase P gene. Compared to the use of animal cells or bacteria, it is also possible to increase the level of amino pear butidase P production ability by using yeast. An example of expressing the aminopeptidase-P gene in yeast is described below.

Yeast - Escherichia coli shuttle vector pAT
77, pAM82 was developed by Miyashita et al. (Proc. Natl.
, Acad.

Set, 'USA 801-5 (1983)). Vector pAM82 is pAT77
are derivatives of , and both have arsl markers (
Stinehcomb DTet al Nature
282.39-43 (1979), 2 μmol
(Broach J, R, at al, Geue,
8. 121-133 (1979).

leu 2 (Ratzkln B, at a
l Proc, Natl, Aead.

';ci, USA 74.474-491 (1979
)), yeast acid phosphatase (APase)
has a promoter for Both vectors carry ampicillin resistance marker (APγ) pBR322
It has a DNA fraction consisting of 3,700 bases and an amplified origin. The APaae promoter is induced only when a high concentration of phosphate is reduced to a low concentration in the culture medium. In order to express the aminopezotidase P gene, pA
After treating the Pst I-Kpn I fragment of PPO4 with the Flenow fraction of Escherichia furi or T4 DNA polymerase, this fragment and Xho I
The pAM82 cut with Escherichia coli was ligated with the Flenou fragment of Escherichia coli or the one treated with T4 DNA polymerase, and a hybrid plasmid having aminopeptidase-P coding sequence downstream of the yeast APase 7' locomotor sequence was created. Cloning and selection using Koli.

Next, the obtained cilasmid pAM82pAPP was introduced into Satucharomyces cerevisiae AH22 as follows.

Cells of Satucharomyces cerevisiae AH22 (leu
2, his 4, can 1, Cir”
) is cultured aerobically at 30°C with shaking in 1001dYPD medium (100d yeast extract, 2% polypeptone, 2% glucose, PH5.3) in a 500d shake flask. Mid-exponential growth phase (absorbance 0.8 at 610 nm)
Collect cells by centrifugation at 350 rpm for 5 minutes.
TE buffer (1mM) Lis-HCl, pH 7,5,0,
Wash once with 1mM EDTA) and 5mM of the same buffer.
Suspend in l. Take 0.5M of this cell suspension in a test tube and add 0.2M C) f3COOLI or 1.
The same amount of 0 M CsC1 was added and the suspension was incubated at 30 °C.
Shake for 1 hour. Cells in suspension were collected by centrifugation at 3500 rpm for 5 min, washed once with 1 M sorbitol, and treated with Zymolyase 5000 rpm.
Solution (1M sorbitol, 0.1M citrate buffer-1
Resuspend in 1 ml of 0 mM EDTA-Q, 4 mQ/1 nl Zymolyase 5000). Protoplasts are prepared by gently shaking this suspension at 37° C. for 1 hour. The protoplasts were treated with IM sorbitol.
Wash twice, IM lnrubito #-10mM CaC22
-10mM) Lis-HCl solution 1. Ornl (p
Resuspend in H7,5). Plasmid DNA (pAM
82pAPP) is added to the protoplast suspension at 20 μ97 ml and the mixture is incubated for 5 minutes at room temperature. Next, 40%? Liethylene glycol 4000-10mM CaCl2-10mM
) 5 ml of lithium-hydrochloric acid solution (pH 7.5) was added to this mixture, and the protoplasts were precipitated by 10-minute wave centrifugal separation.
-101TLM) Lis-hydrochloric acid solution (pH 7,5) 5
Resuspend in ml. A 0.2 ml portion of the suspension was added to 10 m
1 regenerated agar medium plus 0.1M sorbitol, 0.6
7 Chidayfuco East Nitrogen Base.

2 Tiglucose/amino acid mixed solution (L-glutamic acid, L-asnocragic acid, L-alanine, L-proline, L-threonine, L-serine, L-) IJ 7'
) Fan, L-isoleucine, L-valine.

L-phenylalanine, L-tyrosine, L-methionine, glucine, L-glutamine, L-asparagine, r”
-') gin, L-cystine, L-histidine and L
- a minimum agar plate consisting of arginine (containing 5 m9/dl each) and 2 T agar) (pH 5,3);
Select the “Leu” transformant.

This transformant, namely Satucharomyces cerevisiae AH22 containing pAM82pAPP (pAM82ADF-
1/AH22) was aerobically mixed with histidine (20μ
100 ml of high-phosphate Burkh supplemented with
old) minimal medium (20,0 g/l glucose,
2.0 g/l Asuno! Ragin 1, 51/dl KH
2PO4,2,01/l (Su,)2So4.0.5Scrap M,5o4-7H20,0,33171CaCl2”2
H20, 0.1 m9/lKl, 0.25 m9/l
FeSO4”7H2010,04m9AMnSOa Part 4
H20, 0.02m9/l (NH,)6MO7024
-4T (20,0,60■/l H3BO3,0,04
■4C mouth S04・5H20, 0,31ηll ZnSO
4'7H20, 0.20mli/l thiamine hydrochloride.

i o, o wi inositol, 0.2 m) Calcium guntothenate, 0.2 m9/l pyridoxine hydrochloride, 0.05 mgt7fnl /j aminobenzoic acid, 9.20 m971 nicotinic acid and 2.0 gamma/
The cells are cultured in a pH 5.3 solution containing l-pyotin. When the cell density reached an absorbance of 0.4 at 610 nm, samples were removed and centrifuged in low phosphate minimal medium (20,0
V′l glucose, 2.01/l asnoglagin, 1
．． 5 Fl/l KCl, 2.0 E/1 (NH4
)2so4. o, 5 g/l MgSO4・7H2
0,0,33,9,'1CnC12-2H20,0,1
m9/l KI, 0.25m9/l FeSO4-
7H20, 0,04,'n9/l MnSO4"4H2
0,0,02m9/1(NH4)6Mo7024'4H
20,0,6”9/l H3BO3,0,04mQ/l
CuSO4・5H20,0,31,WI ZnSO4
'7H20, 0.2Wl Thiamine Hydrochloride, 10.0
m/j/l inositol.

0.2 mq/l calcium pantothenate, 0.2
m9/l pyridoxine hydrochloride, 0.05■4-2-ra aminobenzoic acid.

9.2・Nicotinic acid and 2. Induction is carried out by suspending in 0 ml of Or/l biotin, pH 5, 3)l. Control samples are treated in the same way except that they are not transferred to low phosphate medium midway through. When the cell density at 610 nm reaches an absorbance of 0.8, centrifuge 100 M samples of induced and near-induced cultures.

The obtained cells were sieved with Zymolyase 500 (100%).
) 1.2M Sorbitol, 50mM IJ phosphate (p
H7,2') and 14 mM 2-mercaptoethanol, and the suspension was incubated at 30 °C for 30 min.
Ink and bake for a minute. The zorodoplasts are collected by centrifugation and separated in 1 mM of 0.1 tidriton Isolate (1ysi
te) is clarified by centrifuging 1 l at 1000 rpm for 20 minutes. These Escherichia coli or Hasaccharomyces cerevisiae cell extracts were subjected to ammonium sulfate precipitation fractionation.
Perform ion exchange chromatography such as DEAE-Sephadex, and further perform DgAE-5PW + Ph
A purified sample of aminobezotidase P is obtained by performing high performance liquid chromatography such as enyl-5PW.

As described above, by using the microorganism of the present invention, the conventionally known Escherichia coli aminopeptidase-P
This method is advantageous in the separation and purification of aminobutidase-P because the production amount per unit protein is extremely high compared to when producing bacteria are used.

In addition, aminopeptidase-P is used because the substrate specificity of this enzyme is that it specifically cleaves the end of X of X -Pro-... to release the peptide Pro-... N of a protein peptide whose terminal end is proline
It can be used to align the ends.

For example, lefunbinan (produced in Escherichia coli) is used to align the ends of BSF-2ON (Japanese Patent Application No. 61-302699).

Example 1゜Escherichia coli HBOI (Boyer, H.
W, etal, Mo1. Biol, 41.4
59 (1969)) as the original strain for producing aminobezotidase P, we isolated the DNA fragment carrying the genetic information involved in aminopeptidase-P production using the following method, and newly obtained a significant amount of aminobezotidase P. A bacterium that produces peptidase-P was constructed.

(1) Preparation of chromosomal DNA Escherichia coli HBOI 40 (1!) L medium (
Trijiton 1 t, yeast extract 0.5%, NaCto, 5
Excellent, glucose 0.1%, adjusted to pH 7.2) at 37°C
After culturing with shaking for about 3 hours, the cells in the logarithmic growth phase were harvested and then subjected to the usual DNA extraction method using the phenol method (H.

5aito and K, Miura, Bloch
im, Biophys, Acta.

Chromosomal DNA by 72, 6), 9 (1963)
was extracted and purified to obtain a final 20η.

(2) Insertion of chromosomal DNA fragment into vector Add 10 μl each of the chromosomal DNA obtained in (1) and vector pBR322 (commercially available from Takara), add one type of restriction endonuclease, Pit I to each at 37°C. Let time work D
The NA chain was cleaved. Next, each was treated with phenol and chloroform, then ethanol precipitated and collected, and both DNA fragments were treated with 1 mM ATP, 10 mM sothiothreitol,
6.6 rn) J (66mMTris containing MgC62
-T4DNA in HCIs pH 7,5! 77!
The mixture was mixed with 100 units of /-ze (Takara), and the mixture was reacted day and night at 4°C.

(3) Preparation of competent cells Escherichia coli DH1 (Low, B, Proc
.

Natl, Acad, Sci, 60.160 (1
968)) 50! nl! The culture solution was cultured with shaking at 30°C until the absorbance at 650 mμ of the culture solution reached approximately 0.4 to 0.5. After culturing, drain the culture solution to 0.
℃ for 20 minutes, then the bacterial cells were collected by centrifugation, suspended in 25 ml of 50 mM Ca C42 solution, and incubated at 0℃.
After leaving for 1 hour, the bacterial cells were collected by centrifugation and diluted with 50mM
CaC62 and 20% glycerol solution 15tA! The cells were resuspended, and each 0.5 d of the obtained cell suspension was placed in an EsoR-Hundorff tube and stored frozen at -70°C.

(4) Transformation and acquisition of transformed strains Add the DNA solution prepared in (2) to 200μ of the competent cell suspension prepared in (3), leave at 0°C for 30 minutes, and then incubate at 42°C for 2 minutes. After applying a heat shock, the cells were again left at 0°C for 30 minutes to allow the DNA to be incorporated into the cells.

Next, L medium was added IM to this suspension, and after culturing at 37°C for 1 hour, tetracycline was added at 20μji/rnl.
The mixture was spread on an agar plate containing a medium containing the following, and a portion was incubated at 37°C. The colonies that appeared were then smeared onto agar plates containing 25 µji/ml of ampicillin, and a portion of the colony was incubated at 37°C to test for growth. p as a candidate method for assaying aminopeptidase P production in colonies that do not
ick+jp.

(5) Assay for aminopeptidase-P production (4)
Approximately 1,000 colonies of the test candidate method were inoculated (using a 96-well plate) into 300 μl of a medium containing 20 μg of tetracycline and 7 ytt.
The cells were cultured at 37°C for 24 hours. Add 3 mM Gly-Pro-/Naphthylami to 50 μl of this culture solution.
Add 50 μm of Proline (dissolved in 20mM Tris-MC6, pH 8,0), and add 1ml of Proline.
50 μl (0.1 unit) of opeptidase was added and reacted at 37° C. for 4 hours. Add 50% to this reaction solution.
μl of Fast Garnet GBCSol (1
mVIMIn 1M acetate buffer, p+(4,0+1
0% TritonX-100 solution) was added, and azo dye (
The amount of red) produced was measured by absorbance at 550°C. As a result, a candidate method was obtained that was about 10 times more active than the original strain HB],01 control.

Regarding this candidate method, the following experiment was conducted to further examine the enzyme activity at the cell-free extract level.

1 medium containing 20 μg 7Δa of tetracycline
The cells were inoculated into a Sakaro flask containing 00- and cultured with shaking at 37°C. After the culture was completed, the cells were collected, suspended in a 20 mM Trim-HCt, pH 8.5 solution containing 30 μM MnC62, and incubated at 0°C. The extract was obtained by sonication and centrifugation. Using this cell extract, aminopeptidase was extracted as follows.
The activity of P was measured.

20 mM Tris-HCt, pH 8.0, 0.7
1 nl and Q of cell extract, l ml and 2 mM Gl
y-Pro-/Naphthylamide Q, l
ml and reacted at 37°C for 10 minutes.

After stopping the reaction by heat treatment at 100°C for 2 minutes, 0.1 unit of 0.3 units of proline iminobezotidase derived from Bacillus coagulans (Gyudon Yakuhin) was added, and then heated at 37°C for 30 minutes. It reacted for minutes. and 1M acetate buffer, pH 4.0 and 10% Triton X.
Fast G at a concentration of 1 Ginze in a solution consisting of -100
5 ml of a reagent containing arnet GBC was added to develop color. This redness was measured by absorbance at 550°C.

As a result, as shown in Table 1, an approximately 8-fold increase in specific activity was observed compared to the original strain HBIOI and host strain DHI K.

This recombinant strain was named pAPPo 1/I)Hl and has been deposited as FERM-9827.

The unit is still It is expressed as

Table (6) Restriction enzyme map of the inserted DNA part of pAPP old Plasmid pAPP01 containing a DNA fragment carrying the genetic information involved in aminobezotidase P production was prepared by a conventional method (T, Maniae 8 et a
l,・, Molecular Cloning, p
93 (1982), Cold Spring Har
bor Laboratory).

When a restriction enzyme map was prepared by cutting with a restriction enzyme that mainly recognizes a 6-nucleotide sequence, it was found that the protein had a cleavage site as shown in FIG.

Furthermore, the inserted portion excised with Pstl was approximately 4 kb in size.

Example 2 Next, the following study was conducted to shorten the 4 kb inserted fragment of pAPPOl.

First, a 4 kb Pst I fragment was excised from pAPPo 1, and the Pst I fragment of cilasmid puc 19 was cut out.
pAPPo2 inserted into the site was constructed (Figure 2).

Next, cut pAPPo2 with Kpn l to approximately 1.7
kb and 7.1 kb fragments were prepared. That is, pAPPo220 μy was cut with the restriction enzyme Kpn I, and the reaction solution was diluted with 0.9% containing etizome promide.
1.7kb and 7.1k by agarose gel electrophoresis
The two fragments of b were separated and the dal of each was cut out. Each of these dals was placed in a dialysis tube and electrically eluted into 90mM Trig-borate buffer pH 7.5 in the dialysis tube, treated with phenol and chloroform, and 1.7kb and 7.5kb were precipitated with ethanol.
0.8I for each 1 kb fragment! l and 5μ
y collected.

Next, as shown in FIG. 2, the K 7.1 kb fragment was subjected to an IT ligation reaction to obtain pAPPo3. pAPPO3/ transformed with this pAPPo3
When strain JM83 was assayed for aminobezotidase P activity using the method described in Example 1, it showed only the same activity as the therapy (Table 2).

For the 1.7 kb fragment, this fragment was converted into the same (Kpn of pUc19 (Takara, commercially available)
It was inserted into the I site by ligation reaction to obtain pAPPo4.

pAPPO4/JM transformed with this pAPPo4
As shown in Table 2, strain 83 has high aminoiptidase P.
It exhibited specific activity approximately 40 times that of therapeutic HBIOI. Based on these facts, the DNA fragment that carries the genetic information involved in aminopeptidase-P production is this one.
．． It was revealed that it exists in a 7 kb Kpn I-Pst I fragment. By using a pUC vector capable of replicating to high copy numbers in Escherichia coli, we succeeded in constructing a pAPPO4/JM83 strain that produces a significant amount of aminopeptidase-P. This strain has been deposited as FERM-9828.

Table 2 Example 3゜Next, Kpn I - Pst I of pAPPo4 1.7
The base sequence of the kb fragment was determined. The base sequence is from M13 phage (J, Messlng at at,
dideoxynucleotide chain termination method (F., Gene, 19.269 ('82)).

Sanger et al., Proc., Natl.
, Aead, Sc1. U, S, A+74, 546
3 ('77)). The determined nucleotide sequence and amino acid sequence are shown in Figure 3. The amino acid sequence determined here is pAPPo constructed in Example 2.
This result matched the 20 N-terminal amino acid partial sequences determined using aminopezotidase P protein purified by culturing the 4/JM83 strain.

[Brief explanation of the drawing]

Figure 1 shows the restriction enzyme map of the aminopeptidase-P gene in pAPPOl, Figure 2 shows the construction of pAPPO4 from pAPPOl, and Figure 3 shows the entire base sequence of the aminopeptidase-P gene region and the amino acid sequence of aminopeptidase-P.

Claims (1)

[Scope of Claims] (1) A gene encoding a polypeptide having aminopeptidase-P activity. (2) The first gene has the following base sequence.
Gene according to claim. [There is a gene sequence] (3) The gene is 1 in the base sequence stated in the second claim.
~ The first sequence base that starts from the third ATG sequence and has a sequence base from the ATG sequence to at least the 1321st to 1323rd CAA sequences in the base sequence.
Gene according to claim. (4) The gene is 4 in the base sequence according to the second claim.
- The first sequence base starting from the 6th AGT sequence and having a sequence base from the AGT sequence to at least the 1321st to 1323rd CAA sequences in the base sequence.
Gene according to claim. (5) The gene is 1 in the base sequence according to the second claim.
The gene according to claim 3, which has a base sequence ending with the 321st to 1323rd CAA sequence. (6) The gene is 1 in the base sequence according to the second claim.
The gene according to claim 4, which has a base sequence ending with the 321st to 1323rd CAA sequence. (7) The gene is 1 in the base sequence according to the second claim.
The gene according to claim 3, which has a base sequence ending with the 324th to 1326th TGA sequence. (8) The gene is 1 in the base sequence according to the second claim.
The gene according to claim 4, which has a base sequence ending with the TGA sequence at positions 324 to 1326. (9) The gene is 1 in the base sequence according to the second claim.
The gene according to claim 1, which has a sequence base starting from A at position A and ending at A at position 1323 in the base sequence. (10) The gene according to claim 1, wherein the gene has a sequence base starting from the 4th A in the base sequence according to claim 2 and ending with A at the 1323rd position in the base sequence. (11) The gene according to claim 1, wherein the gene has a sequence base starting from G at position -367 in the base sequence according to claim 2 and ending at C at position 1483 in the base sequence from said G. . (12) The gene according to claim 1, wherein the gene has a base sequence corresponding to the following amino acid sequence (I). (13) The gene according to claim 1, wherein the gene has a base sequence corresponding to the following amino acid sequence (II). [There is a gene sequence] (14) Consists of a gene encoding a polypeptide having aminopeptidase-P activity and a vector DNA that can be replicated in prokaryotic cells or eukaryotic cells, and the coding sequence of the gene is A recombinant DNA characterized by being located downstream of a promoter sequence. (15) The recombinant DNA according to claim 14, wherein the gene has the base sequence according to claim 2. (16) The gene starts from the first to third ATG sequences in the base sequence according to the second claim, and from the ATG sequence at least 1321 to 1323 in the base sequence.
15. The recombinant DNA according to claim 14, which has a sequence base up to the th CAA sequence. (17) The gene starts from the 4th to 6th AGT sequence in the base sequence according to the second claim, and from the AGT sequence at least 1321 to 1323 in the base sequence.
15. The recombinant DNA according to claim 14, which has a sequence base up to the th CAA sequence. (18) The recombinant DNA according to claim 16, wherein the gene has a base sequence ending with the 1321st to 1323rd CAA sequence in the base sequence according to claim 2. (19) The recombinant DNA according to claim 17, wherein the gene has a base sequence ending with the 1321st to 1323rd CAA sequence in the base sequence according to claim 2. (20) The recombinant DNA according to claim 16, wherein the gene has a base sequence ending at the 1324th to 1326th TGA sequence in the base sequence described in claim 2. (21) The recombinant DNA according to claim 17, wherein the gene has a base sequence ending at the 1324th to 1326th TGA sequence in the base sequence described in claim 2. (22) The recombination according to claim 14, wherein the gene has a sequence base starting from the first A in the base sequence according to claim 2 and ending with A at the 1323rd position in the base sequence. DNA. (23) The recombinant according to claim 14, wherein the gene has a sequence base starting from the 4th A in the base sequence according to claim 2 and ending with A at the 1323rd position in the base sequence. DNA. (24) The set according to claim 14, wherein the gene has a sequence base starting from G at position -367 in the base sequence according to claim 2 and ending at C at position 1483 in the base sequence from said G. Replacement DNA. (25) The gene comprises the amino acid sequence according to claim 12 (
The recombinant DNA according to claim 14, which has a base sequence corresponding to I). (26) The gene comprises the amino acid sequence according to claim 13 (
15. The recombinant DNA according to claim 14, which has a base sequence corresponding to II). (27) The recombinant DNA according to claim 14, wherein the prokaryotic cell line belongs to the genus Escherichia. (28) The recombinant DNA according to claim 14, wherein the prokaryotic cell line belongs to Escherichia coli. (29) The recombinant DNA according to claim 14, wherein the eukaryotic cell line belongs to the genus Saccharomyces. (30) The recombinant DNA according to claim 14, wherein the eukaryotic cell line belongs to Saccharomyces cerevisiae.
. (31) Consists of a gene encoding a polypeptide having aminopeptidase-P activity and a vector DNA capable of replicating in prokaryotic cells or eukaryotic cells, and the coding sequence of the gene is located downstream of the promoter sequence. A eukaryotic or prokaryotic cell transformed with a certain recombinant DNA. (32) The cell according to claim 31, wherein the gene has the base sequence according to claim 2. (33) The gene starts from the 1st to 3rd ATG sequences in the base sequence according to the second claim, and from the ATG sequence at least 1321 to 1323 in the base sequence.
32. The cell according to claim 31, having a sequence base up to the th CAA sequence. (34) The gene starts from the 4th to 6th AGT sequence in the base sequence according to the second claim, and from the AGT sequence at least 1321 to 1323 in the base sequence.
32. The cell according to claim 31, having a sequence base up to the th CAA sequence. (35) The cell according to claim 33, wherein the gene has a base sequence ending with the 1321st to 1323rd CAA sequence in the base sequence according to claim 2. (36) The cell according to claim 34, wherein the gene has a base sequence ending with the 1321st to 1323rd CAA sequence in the base sequence according to claim 2. (37) The cell according to claim 33, wherein the gene has a base sequence ending at the 1324th to 1326th TGA sequence in the base sequence described in claim 2. (38) The cell according to claim 34, wherein the gene has a base sequence ending at the 1324th to 1326th TGA sequence in the base sequence described in claim 2. (39) The cell according to claim 31, wherein the gene has a sequence base starting from the first A in the base sequence according to claim 2 and ending with the 1323rd A in the base sequence. (40) The cell according to claim 31, wherein the gene has a sequence base starting from the 4th A in the base sequence according to claim 2 and ending with A at the 1323rd position in the base sequence. (41) The cell according to claim 31, wherein the gene has a sequence base starting from G at position -367 in the base sequence according to claim 2 and ending at C at position 1483 in the base sequence from said G . (42) The gene comprises the amino acid sequence according to claim 12 (
32. The cell according to claim 31, which has a base sequence corresponding to I). (43) The gene comprises the amino acid sequence according to claim 13 (
32. The cell according to claim 31, which has a base sequence corresponding to II). (44) The cell according to claim 31, wherein the prokaryotic cell belongs to the genus Escherichia. (45) The cell according to claim 31, wherein the prokaryotic cell belongs to Escherichia coli. (46) The cell according to claim 31, wherein the eukaryotic cell belongs to the genus Saccharomyces. (47) The cell according to claim 31, wherein the eukaryotic cell belongs to Saccharomyces cerevisiae. (48) Consists of a gene encoding a polypeptide having aminopeptidase-P activity and a vector DNA capable of replicating in prokaryotic cells or eukaryotic cells, and the coding sequence of the gene is located downstream of the promoter sequence. A method for producing aminopeptidase-P, which comprises culturing eukaryotic or prokaryotic cells transformed with a certain recombinant DNA in a medium, and collecting the produced aminopeptidase-P. (49) The method according to claim 48, wherein the gene has the base sequence according to claim 2. (50) The gene starts from the first to third ATG sequences in the base sequence according to the second claim, and from the ATG sequence at least 1321 to 1323 in the base sequence.
49. The method according to claim 48, wherein the method has a sequence base up to the th CAA sequence. (51) The gene starts from the 4th to 6th AGT sequence in the base sequence according to the second claim, and from the AGT sequence at least 1321 to 1323 in the base sequence.
49. The method according to claim 48, wherein the method has a sequence base up to the th CAA sequence. (52) The method according to claim 50, wherein the gene has a base sequence ending with the 1321st to 1323rd CAA sequence in the base sequence described in claim 2. (53) The method according to claim 51, wherein the gene has a base sequence ending with the 1321st to 1323rd CAA sequence in the base sequence described in claim 2. (54) The method according to claim 50, wherein the gene has a base sequence ending at the 1324th to 1326th TGA sequence in the base sequence described in claim 2. (55) The method according to claim 51, wherein the gene has a base sequence ending at the 1324th to 1326th TGA sequence in the base sequence described in claim 2. (56) The method according to claim 48, wherein the gene has a sequence base starting from the first A in the base sequence according to claim 2 and ending with the 1323rd A in the base sequence. (57) The method according to claim 48, wherein the gene has a sequence base starting from the 4th A in the base sequence according to claim 2 and ending with A at the 1323rd position in the base sequence. (58) The method according to claim 48, wherein the gene has a sequence base starting from G at position -367 in the base sequence according to claim 2 and ending at C at position 1483 in the base sequence from said G. . (59) The gene comprises the amino acid sequence according to claim 12 (
49. The method according to claim 48, which has a base sequence corresponding to I). (60) The gene comprises the amino acid sequence according to claim 13 (
49. The method according to claim 48, which has a base sequence corresponding to II). (61) The method according to claim 48, wherein the prokaryotic cell belongs to the genus Escherichia. (62) The method according to claim 48, wherein the prokaryotic cell belongs to Escherichia coli. (63) The method according to claim 48, wherein the eukaryotic cell belongs to the genus Saccharomyces. (64) The method according to claim 48, wherein the eukaryotic cell belongs to Saccharomyces cerevisiae.